I. Pseudorabies Disease
Pseudorabies, a highly contagious disease of swine and other livestock, such as cattle, sheep, and goats, is caused by Herpesvirus suis (hereinafter "pseudorabies virus" or "PRV"). In swine, the disease causes respiratory illness and encephalitis which may progress to death. Other common consequences of infection in swine are abortions, neonatal demise, reduced litter size, and slower growth rates. In other livestock, most notably cattle, PRV infection almost invariably proceeds to a lethal encephalitis.
Pseudorabies has become a major threat and cause of economic loss to the swine industry throughout the world. There is also considerable alarm over the spread of pseudorabies to cattle and other farm animals. Within the last ten years, economic losses have escalated because of the emergence of more virulet strains of PRV and the widespread dissemination of the disease. Today, it is estimated that 8.0% of the 80 million hogs on farms in the United States are infected, in comparison to less than 0.8% a decade ago.
The clinical symptoms and consequences of PRV infection may be moderated or prevented by the use of vaccines comprising either killed or modified live, i.e., attenuated strains of PRV. However, most existing vaccines have failed to control the spread of pseudorabies disease because of a unique biological property of PRV and the other alpha herpesviruses, such as herpes simplex virus types 1 and 2 (hereinafter "HSV-1" and "HSV-2", respectively), varicella-zoster, infectious bovine rhinotracheitis virus, marmoset herpesvirus, and equine herpesvirus type 1.
More specifically, alpha herpesviruses, have the special ability to enter into a dormant state in neural tissues. That is, as an animal recovers from the initial generalized infection, alpha herpesviruses retreat to portions of the nervous system where they become quiescent and impervious to the body's immune defenses. This dormant infection, i.e., latency, may be unexpectedly reactivated, resulting in recrudescence of disease or in a contagious condition known as the carrier state, wherein the infected animal shows no outward symptoms of the disease but can transmit or "shed" infectious alpha herpesviruses intermittently, so as to cause the spread of infection and epidemic outbreaks.
II. Known Modified Live Virus PRV Vaccines
Previously, modified live virus PRV vaccines have been produced by multiple passages of the virus in chick and/or monkey tissue culture cells (see: Skoda, R., Brauner, I., Sadecky. E., and Mayer V., Acta Virol. 8:1-9 (1964) and Bartha, A., Magy. Allatrov. Lapja 16:42-45 (1961)). During tissue culture passages, mutations accumulate as the virus adapts to its new environment. These undefined mutations adversely affect virus reproduction in the natural host, resulting in virus attenuation.
A problem with the above-described modified live virus PRV vaccines is that the animal often becomes a carrier of the dormant vaccine virus. As a result, usage of these vaccines can result in two undesirable situations which impede their safety and effectiveness. First, abortions, stillbirths, and fatal infections in newborns can be caused by some vaccine viruses as they are shed by vaccinated carriers. Second, the repeated circulation of vaccine virus within a herd can result in a reversal of the process of attenuation such that the vaccine virus reverts to the pathogenic parent strain. Under such circumstances; widespread vaccination will undesirably promote the dissemination of the disease.
In addition to the above-described disadvantages, the previously known PRV vaccines, while substantially minimizing symptoms of illness, do not prevent the animal from acquiring a dormant infection with pathogenic field strains. Thus, despite vaccination, an animal may become a carrier of the disease and transmit it to susceptible animals. These carriers of the disease, when moved between farms and market, will shed not only the dormant vaccine virus as discussed above, but also the disease virus. This results in the undesirable transmission of the disease across geographic barriers and state boundaries.
In order to overcome the above-described disadvantages, temperature-resistant pseudorabies viruses which fail to produce a functional thymidine kinase (hereinafter "TK") enzyme as a result of either a mutagen-induced mutation or a deletion in the thymidine kinase gene (hereinafter "tk gene") were developed (see: U.S. Pat. No. 4,514,497, which U.S. patent is incorporated by reference herein in its entirety).
Nonetheless, the dormancy feature of PRV makes it difficult to effect eradication of the disease through the application of quarantine measures which are intended to prevent the spread of the disease by the isolation of infected herds and the slaughter of infected animals. That is, with existing vaccines it is difficult to determine whether a specific animal, which does not show symptoms of illness, is a carrier of a dormant PRV since usage of most current vaccines mask infections. Hence, since animals which appear healthy may actually be carriers and thus spreaders of PRV, it is important to be able, even after vaccination, to identify infected animals and herds so as to be able to apply quarantine measures. Embodiments of the present invention were developed in order to meet this need.
Moreover, Federal regulations require that swine intended for interstate movement for market or resale must be tested and shown not to be carriers of PRV (i.e., sero-positive for PRV). With all current killed and modified live pseudorabies virus vaccines, a producer who is forced by the circumstances of PRV infection within his herd to vaccinate the susceptible animals finds himself in a position of severe economic disadvantage since the vaccination of the entire stock will result in a positive serological test for PRV. In addition, revaccination to enhance protection will further increase PRV antibody titers. As a result, the farmer's ability to sell his stock is severely restricted.
A vaccine that can safely be administered, protects livestock from disease and dormant infections caused by field strains of PRV, and yet, does not produce a positive test for PRV would allow vaccination programs to be pursued unhindered by the fear of quarantine. The producer could then minimize losses within his own herd, while the animal health authorities could continue with effective, and no longer excessive, control measures. Embodiments of the present invention are also developed in order to meet these needs.
III. The Genomes of PRV Strains
The genomes of PRV strains consist of linear, double-stranded, noncircularly permutated DNA molecules, approximately 146 kilobase pairs (hereinafter "Kbp") in size. Analyses of the genomes of virulent strains of PRV by electron microscopy as well as by restriction nuclease enzymes has shown that they all contain a sequence of DNA, designated as the short unique (hereinafter "U.sub.S ") sequence, about 10 Kbp in size. The U.sub.S sequence is bracketed by inverted repeat and terminal repeat sequences (hereinafter "IR.sub.S " and "TR.sub.S ", respectively), each about 15 Kbp in size (see: FIG. 1). Another unique sequence, i.e., the long unique (hereinafter "U.sub.L ") sequence, which is about 111 Kbp in size, comprises the remainder of the molecule.
The genomes of virulent PRV strains are classified as Class D DNA molecules. This is because the U.sub.S region of the genome, which is bracketed by the IR.sub.S and TR.sub.S regions, is found in two orientations relative to the U.sub.L region. The PRV DNA molecules isolated from such virions contain equimolar amounts of these two isomeric forms. Digestion of the DNA with restriction nucleases that do not cut within the IR.sub.S and TR.sub.S regions (e.g., with BglIII; see: FIG. 1) produce, in addition to the molar fragments originating entirely from the unique regions of the genome, the two half-molar terminal fragments, BglII-D and BglII-E, and, also, two half-molar fragments which span the repeats and the unique sequences (i.e., BglII-A). In FIG. 1, only one BglII-A fragment is shown, but, in fact, there are two fragments of almost the same size which comigrate.
Restriction enzyme cleavage maps of virulent laboratory strains of PRV have been described (see: Ben-Porat, T. and Kaplan, A. S., In: The Herpesviruses, Ed. Roizman, B. (Plenum Press, New York). Vol. 3, pp. 105-173 (1985)).
The restriction maps for the KpnI, BamHI, and BglII fragments for the typical PRV(Kaplan) strain are shown in FIG. 1 (see: Lomniczi, B., Blankenship. M. L., and Ben-Porat, T., J. Virol. 49:970-979 (1984)). The KpnI and BamHI restriction patterns for the virulent Aujeszky strain of PRV (ATCC No. VR-135), which closely resembles that of the PRV(Kaplan) strain are shown in FIG. 3, lanes 3 and 8 of U.S. Pat. No. 4,514,497. Restriction patterns for more than 90 additional field strains of PRV isolated from various regions of the United States, Europe, and Taiwan have been described (see: Gielkins, A. L. J., van Oirschot, J. T., and Berns, A. J. M., J. Gen. Virol. 66:69-82 (1985); Kit; S., Kit, M., Lawhorn, B., and McConnell, S., In: High-Technology Route to Virus Vaccines, Eds. Dreesman. G. R., Bronson, J. G., and Kennedy, R. C. (American Society of Microbiology, Washington, D.C.), pp. 82-99 (1985); Lomniczi, B., Blankenship. M. L., and Ben-Porat, T., J. Virol., 49:970-979 (1984 ); Paul, P. S., Mengeling, W. L., and Pirtle, E. C., Arch. Virol. 73:193-198 (1982); Pirtle, E. C., Wathen, M. W., Paul, P. S., Mengeling, W. L., and Sacks, J. M., Am. J. Vet. Res. 45:1906-1912 (1984); and Pritchett, R. F., Bush, C. E., Chang, T. J. Wang, J. T., and Zee, Y. C., Am. J. Vet. Res. 45:2486-2489 (1984)). Although the restriction patterns are generally similar to those shown for the Aujeszky strain of PRV (see: FIG. 3, lanes 3 and 8 of U.S. Pat. No. 4,514,497), cleavage pattern variations are demonstrable which distinguish different field isolates from one another. One type of variation involves the loss or gain of restriction endonuclease cleavage sites. For example, KpnI fragments of 3.15 Kbp and 10.7 Kbp are found in three Taiwan isolates, but not in the United States and European strains. Instead, the latter strains have a 13.8 Kbp KpnI fragment (KpnI-C), indicating that the Taiwanese strains have an additional KpnI cleavage site in the KpnI-C fragment (see: Pritchett, R. F., Bush, C. E., Chang, T. J., Wang, J. T., and Zee, Y. C., Am. J. Vet. Res. 45:2486-2489 (1984)).
A second type of variation, which occurs at higher frequency for fragments mapping in the IR.sub.S and TR.sub.S regions of the PRV genome, involves sequence additions or deletions from existing fragments. This type of heterogeneity is particularly noticeable for the BamHI-5 or KpnI-K fragment of PRV strains and results from reiteration of sequences in these fragments.
A third type of variation involves minor changes in the size of homologous fragments. Since no fragments are lost and no new fragments are generated that could account for these molecular weight shifts, these variations are believed to result from small additions or deletions in preexisting fragments rather than by the addition or loss of restriction endonuclease cleavage sites.
The various PRV strains that have been studied have been isolated from epizootically unrelated outbreaks of pseudorabies and are highly virulent. This demonstrates that the above-described variations in restriction nuclease patterns are unrelated to PRV viability or virulence.
The restriction nuclease patterns of several attenuated vaccine strains of PRV, isolated after repeated passage in tissue culture, have also been described. Examples of these are the Bartha A57 and K strains, isolated in Hungary, the SUCH and Bucharest(BUK) strains, isolated in Romania (see: Zuffa, A. and Salaj, J., Veterinarni Medicina 17:201-210 (1972)), derivatives of the Bucharest strains, e.g., the Norden, PRV(BUK-5), and PRV(BUK-7) strains (see: Paul, P. S., Mengeling, W. L., and Pirtle, E. C., Arch. Virol. 73:193-198 (1982); U.S. Pat. No. 4,514,487; and Kit. S., Kit, M., and Pirtle, E. C., Am. J. Vet. Res. 46:1359-1367 (1985)), and the NIA-4 strain, a derivative of the virulent NIA-3 strain isolated in Ireland (see: Baskerville, A., McFerran, J. B., and Dow, C., Vet. Bull. 43:465-480 (1973)).
The KpnI and BamHI restriction patterns of the PRV(BUK-5) strain are shown in FIG. 3, lanes 2 and 7 of U.S. Pat. No. 4,514,497. The sizes of the KpnI, BamHI, and BglII restriction fragments of the PRV(BUK-5) and PRV(BUK-7) strains are summarized in Table 1, infra (see: U.S. Pat. No. 4,514,497)).
FIG. 3 of U.S. Pat. No. 4,514,497 demonstrates that the DNA restriction patterns of the virulent Aujeszky strain of PRV and the attenuated PRV(BUK-5) strain differ in the following three ways. First, the attenuated PRV(BUK-5) DNA contains several restriction fragments (e.g., BamHI-10B, BamHI-0, and KpnI-X) which are not present in the DNA of the virulent Aujeszky strain of PRV. These altered fragments in the Bucharest vaccine strains of PRV, such or Norden and PRV(BUK-5), result from the fact that a sequence normally present at the end of the U.sub.L region in all of the virulent PRV strains is also found in inverted form at the junction of the U.sub.L and the IR.sub.S regions in the Bucharest strains. As a result, both the U.sub.L and U.sub.S regions of the Bucharest strains can invert relative to each other to produce four DNA isomers (i.e., Class E DNA) (see: Lomniczi, B., Blankenship, M. L., and Ben-Porat, T., J. Virol. 49:970-979 (1984)).
Second, the BamHI-11 fragment of PRV(BUK-5) migrates more slowly during gel electrophoresis and, hence, is larger in size than the BamHI-11 fragment of the Aujeszky strain of PRV. The BamHI-11 fragment encodes the PRV tk gene and other genes. Nucleotide sequencing studies have demonstrated that the increased size of the PRV(BUK-5) BamHI-11 fragment results from a reiteration of about 200 base pairs (hereinafter "bp") in the noncoding sequence that bridges the PRV tk gene and the next gene downstream from the PRV tk gene. The reiterated sequence contains a polyadenylation (AATAAA) signal (see: Kit, S., Kit, M., and Otsuka, H. In: Herpesvirus, Ed. Rapp, F. (Alan R. Liss, Inc., New York), pp. 311-327 (1984)). This reiterated sequence is not observed in the BamHI-11 fragment of the Aujeszky strain of PRV.
The third difference is especially important. The attenuated Bucharest, Bartha, and NIA-4 vaccine strains of PRV all possess a deletion of approximately 4 Kbp in the KpnI-I fragment. This corresponds to a loss of the entire BamHI-12 fragment and adjacent sequences from the BamHI-7 fragment (see: FIG. 1). This deletion is characteristic of attenuated strains and has not been observed in the many virulent PRV strains which have been isolated in the United States, Europe, and Taiwan. This deletion in the KpnI-I fragment accounts, at least in part, for the reduced virulence of the attenuated Bartha, Bucharest, and NIA-4 PRV strains. Thus, marker transfer experiments which restore all of the KpnI-I sequences of field strains to the attenuated vaccine strains have been found to restore virulence, while the genetic engineering of deletions in this KpnI-I fragment of virulent PRV strains has been found to produce an attenuated PRV (see: Lomniczi, B., Watanabe, S., Ben-Porat, T., and Kaplan, A. S., J. Virol. 52:198-205 (1984); Lomniczi, B., Blankenship, M. L., and Ben-Porat, T., J. Virol. 49:970-979 (1984), Berns, A., van den Ouweland, A., Quint, W., van Oirschot, J., and Gielkens, A. L. J., J. Virol. 53:89-93 (1985); Gielkens, A. L. J., van Oirschot, J. T., and Berns, A. J. M., J. Gen. Virol. 66:69-82 ( 1985); and Pritchett, R. F., Bush, C. E., Chang, T. J., Wang, J. T., and Zee, Y. C., Am. J. Vet. Res. 45:2486-2489 (1984)). The sequences deleted from the attenuated vaccine strains but which are present in virulent PRV field strains encode a minor PRV glycoprotein, designated gI or gA (see: Mettenleiter, T. C., Lukacs, N., and Rziha, H. J., J. Virol. 53:52-57 (1985); and Mettenleiter, T. C., Lukacs, N., and Rziha, H. J., J. Virol. 56:307-311 (1985)). The above-described observations indicate that the products of genes encoded by KpnI-I(BamHI-12+7) sequences of PRV are markers for PRV virulence.
Recently, it has been found that the Bartha K strain of PRV contains a "leaky" mutation in a PRV gene encoding a glycoprotein with an apparent molecular weight of about 92,000 to 98,000 daltons (hereinafter "g92") (see: Paul, P. S., Mengeling, W. L. and Pirtle, E. C., Arch. Virol. 73:193-198 (1982)). That is, g92 glycoprotein is produced at about 10% of its normal levels. This "leaky" mutation may account, in part, for the reduced virulence of the Bartha K strain of PRV. This "leaky" mutation in the PRV g92 gene is not found in the Bartha A57 strain (see: Ben-Porat, T., DeMarchi, J., Pendrys, J., Veach, R. A. and Kaplan, A. S., J. Virol. 57:191-196 (1986)).
It is to be emphasized, however, that all of the conventional attenuated vaccine strains, that is Bucharest and its derivatives, Bartha and its variants, and NIA-4, are unaltered in their tk gene, i.e., they all produce fully functional, virus-specific TK enzyme.
Further attenuation of the above-described PRV strains has been achieved by the isolation of spontaneous or mutagen-induced tk.sup.- viruses, or by the isolation of tk.sup.- deletion mutants of PRV (see: U.S. Pat. No. 4,514,497; Kit, S., Kit, M., and Pirtle, E. C., Am. J. Vet. Res. 46:1359-1367 (1985); and Lomniczi, B., Watanabe, S., Ben-Porat, T., and Kaplan, A. S., J. Virol. 52:198-205 (1984)). Thus, virulence of PRV is multigenic, i.e., virulence of PRV is the result of more than one active gene.
The restriction pattern of a PRV mutant with a deletion in the tk gene, i.e., PRV(BUK-dl 3) (ATCC No. VR-2074), is shown in FIG. 3, lanes 1 and 6 of U.S. Pat. No. 4,514,497. From FIG. 3 therein, it can be seen that the BamHI and KpnI restriction patterns of the tk.sup.- deletion mutant, i.e., PRV(BUK-dl 3), are the same as that of the tk.sup.+ parental strain, i.e., PRV(BUK-5), with the exception that the BamHI-11 fragment, designated dl 11 and the KpnI-J.sub.L fragments, which encode the PRV tk gene (see: FIG. 2, plasmid pBK-J.sub.L), migrate more rapidly and, hence, are smaller by about 150 bp in the PRV(BUK-dl 3) deletion mutant than in the parental PRV(BUK-5) strain (Compare FIG. 3 lanes 2 and 7 with lanes 1 and 6 of U.S. Pat. No. 4,514,497).
IV. Pseudorabies Virus Envelope Proteins
Pseudorabies virus particles are approximately 180 nm in diameter and, like other herpesviruses, comprise an icosahedral capsid (100 nm in diameter) composed of 162 capsomers surrounded by a lipoprotein envelope. Removal of this viral envelope with nonionic detergents, such as Triton X-100 or Nonidet P40, allows the separation of the envelope from the viral nucleocapsid, which contains all of the DNA and about half of the viral proteins. The nucleocapsid comprises three major proteins, about 142,000, 35,000, and 32,000 daltons in size, another protein of about 62,000 daltons in size, and about 12 other minor proteins ranging in size from 10,000 to 115,000 daltons. The envelope contains the remainder of the viral proteins, which include at least seven glycoproteins and a nonglycosylated protein. As with other alpha-herpesviruses, such as HSV-1, the envelope proteins, and their precursors, have a role in inducing cellular and humoral immune responses; they function in virus penetration into infected cells; and they promote virus-cell fusion.
Sedimentation analyses and chromatographic studies of the PRV envelope proteins have revealed that several of them are complexed with one another, with some being covalently linked via disulfide bridges. Further analyses of the envelope proteins by immunoprecipitation with monoclonal antibodies has shown that the viral glycoproteins covalently linked by disulfide bridges are gIIa (molecular weight of about 120,000-125,000 daltons), gIIb (molecular weight of about 68,000-74,000 daltons), and gIIc (molecular weight of about 52,000-58,000 daltons) (see: Hampl, H., Ben-Porat, T., Ehrlicher, L., Habermehl, K. O., and Kaplan, A. S., J. Virol. 52:583-590 (1984); and Lukacs, N., Thiel, H. J., Mettenleiter, T. C., and Rhiza, H. J., J. Virol. 53:166-173 (1985)). All three glycoproteins share extensive sequence homology, as indicated by the identity of their antigenic determinants and by partial polypeptide mapping. Thus, they probably originate from a single precursor protein, i.e., gIIa.
Three minor glycoproteins, i.e., gI (molecular weight of about 115,000 to 120,000 daltons), gIV (molecular weight of about 98,000 daltons), and gV (molecular weight of about 62,000 daltons), form a noncovalently linked complex with p115, i.e., a nonglycosylated protein (molecular weight 115,000 daltons). Another major glycoprotein, i.e., gIII (apparent molecular weight of 92,000 to 98,000 daltons) is not complexed with any other protein.
All of the above-described glycoproteins react with monoclonal antibodies, indicating that they are exposed on the surface of the PRV particles. Further, monoclonal antibodies to gIII inhibit virus adsorption and neutralize virus infectivity in the absence of complement.
Molecular hybridization experiments have been carried out with cloned HSV-1 DNA probes to investigate whether the major HSV-1 glycoprotein genes, i.e., HSV-1 gB, HSV-1 gC, and HSV-1 gD, share nucleotide sequence homology with any of the PRV glycoprotein genes. These studies have revealed that HSV-1 DNA fragments representing HSV-1 gB coding sequences hybridize specifically to PRV DNA which encodes the gII complex, but that HSV-1 DNA fragments representing HSV-1 gC and HSV-1 gD do not specifically hybridize to PRV DNA (see: Robbins, A. K., Gold, C., Enquist, L. W., Whealy, M. E. and Watson, R. J., Abstracts presented at the Tenth International Herpesvirus Workshop, Ann Arbor, Mich. Aug. 11-26, 1985, p. 130). Genetic analyses and studies with monoclonal antibodies have also shown that the PRV gene encoding the gII glycoprotein complex maps at the left end of the U.sub.L region of the PRV genome, in the BamHI-1 fragment, at about 0.110 to 0.128 map units (see: FIG. 1 and Wathen, M., Holland, L., Glorioso, J., and Levine, M. Abstracts presented at the Tenth International Herpesvirus Workshop, Ann Arbor, Mich., Aug. 11-26, 1985, p. 140). This location is close to that of the PRV genes encoding the PRV-specific DNA polymerase and DNA binding proteins. The HSV-1 gB gene is also located next to the HSV-1-specific DNA polymerase and DNA binding protein genes. This further demonstrates the homologous relationship between the PRV gII gene and the HSV-1 gB gene.
The HSV-1 gB gene is the only HSV-1 glycoprotein gene known to have an essential role in viral replication and penetration. That is, temperature-sensitive mutants exist with alterations in the HSV-1 gB gene (see: Spear, P. G. In: The Herpesvirus, Ed. Roizman, B. (Plenum Press, New York), Vol. 3, pp. 315-356 (1985); Holland, T. C., Homa, F. L., Marlin, S. D., Levine, M., and Glorioso, J., J. Virol. 52:566-574 (1984); and Marlin, S. D., Holland, T. C., Levine, M., and Glorioso, J. C., J. Virol. 53:128-136 (1985)). This finding suggests that the PRV gII gene may also be essential for PRV replication.
The gene encoding PRV gI has been mapped in the U.sub.S region of the PRV genome in the BamHI-7 and BamHI-12 fragments (see: FIG. 1). Cell-free translation studies on PRV messenger RNAs transcribed from this region have shown that two polypeptides, with molecular weights of about 78,000 and 83,000 daltons, are transcribed and translated from the BamHI-12 and BamHI-7 fragment. These two polypeptides are precipitated by monoclonal antibodies directed against PRV gI, indicating that the 78,000 and 83,000 molecular weight polypeptides are nonglycosylated gI precursors (see: Mettenleiter, J. C., Lukas, N., and Rhiza, H. J., J. Virol. 53:52-57 (1985)). As indicated previously, the apparent molecular weight of the mature glycosylated gI is about 115,000 to 120,000 daltons.
As discussed above, it has been shown that expression of PRV gI is not obligatory for PRV infectivity, but that gI expression is important for PRV virulence (Mettenleiter, T. C., Lukacs, N., and Rhiza, H. J., J. Virol. 56:307-311 (1985); Berns, A. J. M., van den Ouweland, A., Quint, W., van Oirschot, J. T., and Gielkens, A. L. T., J. Virol. 53:89-93 (1985); Lomniczi, B., Blankenship, M. L., and Ben-Porat, T., J. Virol. 49:970-979 (1984)). That is, three vaccine strains of PRV, namely, Bucharest, Bartha, and NIA-4, lack the BamHI-12 fragment, exhibit deletions in the BamHI-7 fragment, and fail to synthesize gI. The tk.sup.- deletion mutant PRV(BUK-dl 3) also lacks the BamHI-12 fragment and has a deletion in the BamHI-7 fragment. This is as expected since it was derived from the Bucharest strains PRV(BUK-5) and PRV(BUK-7) (see: U.S. Pat. No. 4,514,497 and Kit, S., Kit, M., Pirtle, E. C., Am. J. Vet. Res. 46:1359-1367 ( 1985)). In addition, attenuation of virulent PRV strains has been engineered by making DNA sequence deletions in the BamHI-7 and BamHI-12 fragments of PRV DNA (see: European Patent Publication No. 0141458). On the other hand, most of the virulent laboratory and field strains of PRV contain intact BamHI-7 plus BamHI-12 DNA sequences and therefore express PRV gI. (see: Gielkens, A. L. J., van Oirschot, J. T., and Berns, A. J. M., J. Gen. Virol. 66:69-82 (1985); Pirtle, E. C., Wathen, M. W., Paul, P. S., Mengeling, W. L., and Sacks, J. M., Am. J. Vet. Res. 45:1906-1912 (1984); and Pritchett, R. F., Bush, C. E., Chang, T. J., Wang, J. T., and Zee, Y. C., Am. J. Vet. Res. 45:2486-2489 (1984)).
Cells infected with PRV excrete large amounts of an 89,000 molecular weight sulfated glycoprotein into the extracellular fluid. The sulfated glycoprotein is a nonstructural PRV protein, i.e., it is not a component of PRV particles. Its function is unknown. No major intracellular polypeptides with the same molecular weight as this sulfated glycoprotein have so far been detected. This sulfated PRV glycoprotein has been designated gX, and maps in the U.sub.S region of the PRV genome, in a 2 Kbp DNA fragment which contains parts of BamHI-10 and BamHI-7 fragments (see: Pennington, T. H. and McCrae, M. A., J. Gen. Virol. 34:155-165 (1977); Kerr, C. L. and Pennington, T. H., J. Gen. Virol. 65:1033-1041 (1984); and Rea, T. J., Timmins, J. G., Long, G. W., and Post, L. E., J. Virol. 54:21-29 (1985)). Thus, the PRV gX gene is encoded in the U.sub.S region of the PRV genome in the BamHI-7 plus the BamHI-10 fragments and the PRV gI gene, which is also encoded in the U.sub.S region of the PRV genome, is encoded to the right of the PRV gX gene in the BamHI-7 plus the BamHI-12 fragments (see: FIG. 1).
The region of the PRV genome which encodes for gX has been sequenced and found to include an open reading frame coding for 498 amino acids, flanked by sequences which contain features common to eucaryotic promoters and polyadenylation signals. The predicted molecular weight of the coded amino acid sequence is 53,700 daltons which is considerably smaller than the apparent mass of the 70,000 molecular weight precursor seen after in vitro translation of the gX messenger RNA. This is believed to be due to the high percentage (8.8%) of proline residues in the sequence (see: Rea, T. J., Timmins, J. G., Long, G. W., and Post, L. E., J. Virol. 54:21-29 (1985)).
The gene for yet another PRV glycoprotein, designated gp50, has been mapped at 0.813-0.832 map units, which is at least partly within the same BamHI-7 nucleotide sequences as gX (see: FIG. 1). However, unlike gX, gp50 is present on the surface of PRV particles. gp50 was identified through the isolation of a PRV variant, marl97-1, which was resistant to a neutralizing monoclonal antibody (MCA50-1) directed against wild-type PRV gp50. The marl97-1 mutant is completely resistant to neutralization with the MCA50-1 antibody in the presence or absence of complement, but is neutralized by polyvalent immune sera. The marl97-1 mutant synthesizes and processes gp50 normally, but the mutation prevents the binding and immunoprecipitation of gp50 by the MCA50-1 antibodies. This demonstrates that the mutation is within the structural portion of the gp50 gene affecting the epitope of the monoclonal antibody. The location of the genes for gX and gp50 within the same small region of the PRV genome raises the possibilities that the two glycoprotein genes map very close to each other, or that the glycoproteins are somehow related (see: Wathen, M. W. and Wathen, L. M. K., J. Virol. 51:57-62 (1984)).
A PRV glycoprotein with an apparent molecular weight of 82,000 daltons (hereinafter "gp82") has been mapped in the U.sub.L region of the PRV genome at 0.290 to 0.309 map units on the PRV genome. This is within the BglII-B fragment near the junction of the BamHI-2 and BamHI-1 fragments (see: FIG. 1 and Wathen, M. W., Holland, L., Glorioso, J., and Levine, M. Abstracts presented at the Tenth International Herpesvirus Workshop, Ann Arbor, Mich., Aug. 11-26, 1985, p. 140). gp82 is not essential for replication in cell culture and the absence of gp82 is associated with an altered plaque morphology (syncytial formation). Using monoclonal antibodies raised against gp82, it has been determined that gp82 probably corresponds to g92 (see: Wathen, L. M. K., Platt, V. B., Wathen, M. W., van Deusen, R. A., Whetstone, C. A. and Pirtle, E. C., Virus Research 4:19-29 (1985) and the studies herein).
Finally, the PRV gene encoding the glycoprotein with an apparent molecular weight of about 92,000 to 98,000 daltons i.e., g92, has been mapped at about 0.38-0.42 map units on the PRV genome. This is within the BglII-B fragment near the junction of the BamHI-2 and BamHI-9 fragments (see: FIG. 1 and Robbins, A. K., Weis, J. H., Enquist, L. W., and Watson, R. J., J. Mol. Appl. Genet. 2:485-496 (1984)). It should be noted that this map location is at the opppsite end of the BamHI-2 fragment from the map location assigned to gp82 by Wathen et al (see: Wathen, L. M. K., Platt, V. B., Wathen, M. W., van Deusen, R. A., Whetstone, C. A. and Pirtle, E. C., Virus Research 4:19-29 (1985)).
Nucleotide sequencing studies on the PRV g92 gene have been described (see: Robbins, A. K., Presentations at the Ninth International Herpesvirus Workshop, Seattle, Wash. Aug. 24-29, 1984). These studies have revealed that the DNA fragment spanning the BamHI-2 and BamHI-9 junction contain an open reading frame encoding 479 amino acids. The putative translational start signal is at the NcoI restriction site at 5.2 map units on plasmid pBUK:Stul2/PstI (see: FIG. 4). A putative translational stop codon (TGA) is found 57 nucleotides downstream from the BamHI site connecting fragments BamHI-2 and BamHI-9 (6.6 map units on plasmid pBUK:Stul2/PstI; see: FIG. 4). Thirty-one nucleotides downstream from the TGA stop signal is a consensus "AATAAA" polyadenylation signal.
The molecular weight of the nonglycosylated protein predicted from the g92 sequence is 51,000 daltons. This 51,000 molecular weight polypeptide contains eight potential glycosylation sites, i.e., asparagine-X-threonine or asparagine-X-serine sequences. g92 is believed to represent a mature, processed, and fully glycosylated form of a 51,000 molecular weight precursor. A partially glycosylated precursor of PRV g92, with an apparent molecular weight of about 74,000 to 79,000 daltons, (hereinafter "g74") has also been observed through the use of the drug monensin, which inhibits glycoprotein processing (see: Lukacs, N., Thiel, H. J., Mettenleiter, T. C. and Rziha, H. J., J. Virol 53:166-173 (1985)).
Antisera which specifically reacts, in immunoprecipitation and Western blot analyses, with both g92 and g74, have been obtained by immunizing rabbits with a denatured g74 polypeptide excised after sodium dodecyl sulfate-polyacrylamide gel electrophoresis assays (SDS-PAGE) (see: Robbins, A. K., Weis, J. H., Enquist, L. W., and Watson, R. J., J. Mol. Appl. Genet. 2:485-496 (1984)). In contrast, antisera raised in rabbits against the denatured gll0-g92 group of PRV proteins isolated from SDS-PAGE gels reacts in immunoprecipitation and Western blot analyses predominantly with proteins of apparent molecular weights of 110,000, 92,000, and 55,000 daltons. These experiments, and sucrose gradient centrifugation experiments to be described herein, suggest that g92, and its precursor, g74, which map at the BamHI-2/BamHI-9 junction, correspond to glycoprotein gIII (see: Hampl, H., Ben-Porat, T., Ehrlicher, L., Habermehl, K. O. and Kaplan, A. S., J. Virol. 52:583-590 (1984) and Lukacs, N., Thiel, H. J., Mettenleiter, T. C. and Rziha, H. J., J. Virol. 53:166-173 (1985)).
The production of glycoprotein gIII has been demonstrated in cells infected with virulent PRV strains such as the Rice strain, the Ind-F strain, the Iowa 62-26 strain, the Kaplan strain, the Becker strain and the Phylaxia strain, and with attenuated PRV strains such as the Bartha A57 strain, the Bucharest(Norden) strain and the NIA-4 strain (see: Rea, T. J., Timmins, J. G., Long, G. W. and Post, L. E., J. Virol. 54:21-29 (1985); Wathen, M. W. and Wathen, L. M. K., J. Virol. 51:57-62 (1984); Wathen, L. M. K., Platt, K. B., Wathen, M. W., Van Duesen, R. A., Whetstone, C. A. and Pirtle, E. C., Virus Res. 4:19-29 (1985); Lukacs, N., Thiel, H. J., Mettenleiter, T. C. and Rziha, H. J., J. Virol. 53:166-173 (1985); Robbins, A. K., Weis, J. H., Enquist, L. W. and Watson, R. J., J. Mol. Appl. Genet. 2:485-496 (1984); Hampl, H., Ben-Porat, T., Ehrlicher, L., Habermehl, K. O. and Kaplan, A. S., J. Virol. 56:307-311 (1985)).
As discussed above, the Bartha K strain under-produces glycoprotein g92. That is, the Bartha K strain only produces about 10% of the normal levels of the g92 glycoprotein (see: Ben-Porat, T., De Marchi, J., Pendrys, J., Veach, R. A. and Kaplan, A. S., J. Virol. 57:191-196 (1986)). Nonetheless, this amount of glycoprotein g92 should be sufficient to elicit antibodies to glycoprotein g92 in animals vaccinated with such. As a result, antisera obtained from animals vaccinated with the Bartha K strain should still recognize the same antigens as antisera from pigs vaccinated with other PRV strains. Hence, it should not be possible to distinguish animals vaccinated with the Bartha K strain from animals infected with other PRV vaccines or any PRV field strain. Furthermore, reversion of the Bartha K strain to one which produces normal levels of the g92 glycoprotein is not precluded.
In the present invention, it has been possible for the first time to provide a PRV vaccine, wherein animals vaccinated with such, due to deletion and/or insertion mutations in the g92 gene, do not produce any antigenic polypeptide encoded by the g92 gene and can not revert to the production of g92 antigen. As a result, animals vaccinated with such can be distinguished from animals infected with other PRV vaccines or any PRV field strain, so as to enable the erradication of pseudorabies disease through the application of quarantine measures. Additionally, in the present invention, it has been possible for the first time to provide a PRV vaccine which is both distinguishable from other PRV vaccines and field strains, as discussed above, and which is not only effective in controlling the spread of pseudorabies disease, but wherein the animals vaccinated with such, due to the mutations in the PRV tk gene, are less likely to become a carrier of the vaccine virus and are unlikely to acquire a dormant infection with pathogenic field strains.